Amino acid-producing bacteria and a process for preparing L-amino acids

ABSTRACT

The invention relates to amino acid-producing bacteria from the family Enterobacteriaceae, in particular from the species  Escherichia coli , which contain a stop codon chosen from the group amber, ochre and opal in the nucleotide sequence for the coding region of the rpoS gene and a suppressor for a stop codon chosen from the group amber suppressor, ochre suppressor and opal suppressor. This invention also relates to a process for preparing amino acids, in particular L-threonine, using these bacteria.

[0001] The invention relates to amino acid-producing bacteria of thefamily Enterobacteriaceae, in particular of the species Escherichiacoli, which contain at least one stop codon chosen from the groupcomprising amber, ochre and opal in the nucleotide sequence of thecoding region of the rpoS gene and the corresponding suppressor for thestop codon chosen from the group amber-suppressor, ochre-suppressor andopal-suppressor. This invention also relates to a process for preparingamino acids, in particular L-threonine, using these bacteria.

PRIOR ART

[0002] L-amino acids, in particular L-threonine, are used in humanmedicine and in the pharmaceutical industry, in the foodstuffs industryand very particularly in animal feedstuffs.

[0003] It is known that L-amino acids are prepared by fermentation ofstrains of Enterobacteriaceae, in particular Escherichia coli (E. coli)and Serratia marcescens. Improvements in the methods of preparation arealways being looked for due to the great importance of this process.Process improvements may relate to fermentation technological measuressuch as e.g. stirring and supplying with oxygen, or the composition ofthe nutrient media such as e.g. the sugar concentration duringfermentation, or working up to the product form by e.g. ion exchangechromatography, or the intrinsic performance characteristics of themicroorganism itself.

[0004] To improve the performance characteristics of thesemicroorganisms, the methods of mutagenesis, selection and mutantselection are used. In this way strains are obtained that are resistantto antimetabolites such as e.g. the threonine analogonα-amino-β-hydroxyvaleric acid (AHV) or are auxotrophic for importantregulatory metabolites and produce L-amino acids such as e.g.L-threonine.

[0005] The methods of recombinant DNA engineering have also been usedfor a number of years for the strain improvement of L-aminoacid-producing strains of the family Enterobacteriaceae, by amplifyingindividual amino acid biosynthesis genes and testing the effect onproduction.

[0006] The rpoS gene, which is also known under the name the katF gene,codes for a protein which is called the σ³⁸ factor or σ^(S) factor, theσ³⁸ protein or the σ³⁸ subunit or else the RpoS protein. The followingnames for the rpoS gene are also found in the literature, though lesscommonly: abrD, dpeB, nur, appR, sigS, otsX and snrA. The as factor, asa subunit of RNA-polymerase, controls the expression of a wide varietyof different groups of genes (Loewen et al.; Canadian Journal ofMicrobiology 44 (8): 707-717 (1998)), wherein the regulation mechanismsare often unclear.

[0007] Data on the nucleotide sequence of the rpoS or katF gene can befound in Mulvey and Loewen (Nucleic Acids Research 17 (23): 9979-9991(1989)) and in Blattner et al. (Science 277: 1453-1462 (1997)).Corresponding data can also be found at the National Center forBiotechnology Information (NCBI) of the National Library of Medicine(Bethesda, Md., USA) under the accession numbers X16400 and AE000358.The translation start or the initiation codon for the rpoS gene wasdetermined by Loewen et al. (Journal of Bacteriology 175 (7): 2150-2153(1993)).

[0008] In addition, a number of rpoS alleles are known in strains ofEscherichia coli, for example in strains of the type in W3110 (Ivanovaet al.; Nucleic Acids Research 20 (20): 5479-5480 (1992) and Jishage andIshihama; Journal of Bacteriology 179 (3): 959-963 (1997)).

[0009] In WO 01/05939 it is shown that, after complete switching off ofthe σ³⁸ factor by incorporating a deletion in the rpoS gene of aL-glutamic acid producer, glutamic acid production is improved.

[0010] Nagano et al. (Bioscience Biotechnology and Biochemistry 64 (9):2012-2017 (2000)) report on an Escherichia coli W3110 strain and theL-lysine producing mutants W196 which both contain a rpoS allele whichcontains an amber stop codon (TAG) at the point corresponding toposition 33 in the amino acid sequence of the σ³⁸ protein. Strain W196also contains a mutation in the serU gene coding for L-serine tRNA whichis called supD.

DESCRIPTION OF THE INVENTION

[0011] Whenever amino acids or L-amino acids are mentioned in thefollowing, this is intended to cover all proteinogenic amino acids withthe exception of L-lysine. This means, in particular, L-threonine,L-isoleucine, L-homoserine, L-methionine, L-glutamic acid, L-valine andL-tryptophane, wherein L-threonine is preferred.

[0012] “Proteinogenic amino acids” are understood to be those aminoacids which are constituents of proteins. These include the amino acidsL-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine,L-threonine, L-cysteine, L-methionine, L-proline, L-phenylalanine,L-tyrosine, L-tryptophane, L-asparagine, L-glutamine, L-aspartic acid,L-glutamic acid, L-arginine, L-lysine, L-histidine and L-selenocysteine.

[0013] The invention provides amino acid-producing, in particularL-threonine-producing bacteria from the family Enterobacteriaceae, inparticular from the species Escherichia coli, which 1) contain at leastone stop codon chosen from the group amber, ochre and opal in thenucleotide sequence of the coding region of the rpoS gene and 2) containthe corresponding suppressor for the stop codon, chosen from the groupamber suppressor, ochre suppressor and opal suppressor.

[0014] The bacteria provided by the present invention can produce aminoacids from glucose, saccharose, lactose, fructose, maltose, molasses,optionally starch, optionally cellulose or from glycerine and ethanol.They are representatives of the family Enterobacteriaceae, chosen fromthe genuses Escherichia, Erwinia, Providencia and Serratia. The genusesEscherichia and Serratia are preferred. In the case of the genusEscherichia in particular the species Escherichia coli and in the caseof the genus Serratia in particular the species Serratia marcescens arementioned.

[0015] The L-amino acid-producing bacteria provided by the presentinvention can, inter alia, optionally produce L-lysine as a secondaryproduct in addition to the desired L-amino acid. Bacteria according tothe invention produce at most 0 to 40% or 0 to 20%, preferably at most 0to 10%, particularly preferably at most 0 to 5% of L-lysine comparedwith the amount of desired L-amino acid. This percentage datacorresponds to percentage by weight.

[0016] L-threonine-producing strains from the family Enterobacteriaceaepreferably possess, inter alia, one or more of the genetic orphenotypical features selected from the group: resistance toα-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance toethionine, resistance to α-methylserine, resistance to diaminosuccinicacid, resistance to α-aminobutyric acid, resistance to borrelidin,resistance to rifampicin, resistance to valine analogues such as, forexample, valine hydroxamate, resistance to purine analogues such as, forexample, 6-dimethylaminopurine, a need for L-methionine, optionallypartial and compensatable need for L-isoleucine, a need formeso-diaminopimelic acid, auxotrophy with respect tothreonine-containing dipeptides, resistance to L-threonine, resistanceto L-homoserine, resistance to L-lysine, resistance to L-methionine,resistance to L-glutamic acid, resistance to L-aspartate, resistance toL-leucine, resistance to L-phenylalanine, resistance to L-serine,resistance to L-cysteine, resistance to L-valine, sensitivity tofluoropyruvate, defective threonine dehydrogenase, optionally an abilityto make use of saccharose, enhancement of the threonine operon,enhancement of homoserine dehydrogenase I-aspartate kinase I preferablythe feed-back resistant form, enhancement of homoserine kinase,enhancement of threonine synthase, enhancement of aspartate kinase,optionally the feed-back resistant form, enhancement of aspartatesemialdehyd dehydrogenase, enhancement of phosphoenolpyruvatecarboxylase, optionally the feed-back resistant form, enhancement ofphosphoenolpyruvate synthase, enhancement of transhydrogenase,enhancement of the RhtB gene product, enhancement of the RhtC geneproduct, enhancement of the YfiK gene product, enhancement of a pyruvatecarboxylase, and attenuation of acetic acid formation.

[0017] A stop codon of the amber type is understood to be a stop codonwith the base sequence TAG on the coding strand in a DNA moleculecorresponding to UAG on the mRNA read by this DNA molecule. A stop codonof the ochre type is understood to be a stop codon with the basesequence TAA on the coding strand in a DNA molecule corresponding to UAAon the mRNA read by this DNA molecule.

[0018] A stop codon of the opal type is understood to be a stop codonwith the base sequence TGA on the coding strand in a DNA moleculecorresponding to UGA on the mRNA read by this DNA molecule.

[0019] The stop codons mentioned are also called nonsense mutations(Edward A. Birge: Bacterial and Bacteriophage Genetics (Third Edition),Springer Verlag, Berlin, Germany, 1994).

[0020] The nucleotide sequence for the rpoS gene can be obtained fromthe prior art. The nucleotide sequence for the rpoS gene correspondingto Accession No. AE000358 is given as SEQ ID NO. 1. The amino acidsequence of the relevant RpoS gene product or protein is given in SEQ IDNO. 2.

[0021] The nucleotide sequence for a rpoS allele which contains a stopcodon of the amber type at the point in the nucleotide sequencecorresponding to position 33 in the amino acid sequence of the RpoS geneproduct or protein, corresponding to SEQ ID NO. 1 and SEQ ID NO. 2respectively, is given in SEQ ID NO. 3.

[0022] The concentration of σ³⁸ factor can be determined by thequantitative “Western blot” method as described in Jishage and Ishima(Journal of Bacteriology 177 (23): 6832-6835 (1995)), Jishage et al.(Journal of Bacteriology 178 (18): 5447-5451 (1996)) and Jishage andIshima (Journal of Bacteriology 179 (3): 959-963 (1997)).

[0023] Suppression is generally understood to be the effect whereby theeffects of mutation in a “first” gene are compensated for or suppressedby a mutation in a “second” gene. The mutated second gene or secondmutation is generally called a suppressor or suppressor gene.

[0024] A special case of suppressors relates to alleles of tRNA geneswhich code for abnormal tRNA molecules which can recognise stop codonsso that incorporation of an amino acid takes place instead of chaintermination during translation. Extensive explanations of suppressioncan be found in genetics textbooks such as, for example, the textbook byRolf Knippers “Molekulare Genetik” (6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995) or the text book by Ernst-L. Winnacker “Geneund Klone, Eine Einführung in die Gentechnologie” (Dritter, amendedreprint, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or thetextbook by F. C. Neidhard (Ed.) ,,Escherichia coli and Salmonella,Cellular and Molecular Biology” (2^(nd) Edition, ASM Press, Washington,D.C., USA, 1996).

[0025] The suppressors listed in Tables 1, 2 and 3 are, inter alia, tRNAgenes or alleles or tRNA suppressors known from the prior art which cansuppress stop codons of the amber, ochre or opal type and are thuscalled amber suppressors, ochre suppressors or opal suppressors. Thenames for the particular genes or alleles and suppressors were takenfrom the references cited. TABLE 1 List of amber suppressors Name of thesuppressor Name of the Amino acid gene/allele tRNA incorporated Ref.supD (=Su1) Serine tRNA 2 Ser  1 supE (=Su2, supY) Glutamine tRNA2 Gln 1 supF Tyrosine tRNA 1 Tyr  1 supP (=Su6) Leucine tRNA 5 Leu  1 supU(=su7) Tryptophane tRNA Trp  1 supZ Tyrosine tRNA 2 Tyr  1 t-RNA^(Asp)_(CUA) Asp tRNA (CUA) Lys, Ala, Gln, Arg  2 tRNAPheCUA PhenylalaninetRNA Phe  3 tRNACysCUA Cysteine tRNA Cys  3 suIII+ amber Tyrosine tRNA 1Tyr  4 Su⁺271 Gln/Trp tRNA Gln  5 ARG Arginine tRNA Arg, Lys  6 ARGIIArginine tRNA Arg, Gln  6 LysA20 Lysine tRNA Lys  6 trpT175 TryptophanetRNA Gln  7 Su79 (=trpT179) Tryptophane tRNA Trp  8 tRNACysCUA CysteinetRNA Cys  9 t-RNA^(Asn) _(CUA) Asparagine tRNA Gln 10 Ala2 Alanine tRNAAla 11 Cys Cysteine tRNA Lys 11 ProH Proline tRNA Pro 11 HisA HistidinetRNA His 11 Gly2 Glycine tRNA Gly, Gln 11 Gly1 Glycine tRNA Gly 11 Ile1Isoleucine tRNA Gln, Lys 11 Met (f) Methionine tRNA Lys 11 Ile2Isoleucine tRNA Lys 11 AspM Asparagine tRNA Lys 11 Arg Arginine tNA Lys,Arg 11 Thr2 Threonine tRNA Lys, Thr 11 Val Valine tRNA Lys, Val 11 GluAGlutamic acid tRNA Glu, Gln, Tyr, Arg 11 ECF Phenylalanine tRNA Phe 12ECF9 Phenylalanine tRNA Phe 12 ECF5-2 Phenylalanine tRNA Phe, Thr, Tyr12 ECF10 Phenylalanine tRNA Phe 12 ECF602 Phenylalanine tRNA Phe, Lys,Thr, Val 12 ECF606 Phenylalanine tRNA Phe 12 ECF11 Phenylalanine tRNAPhe 12 ECF12 Phenylalanine tRNA Phe 12 ECF6 Phenylalanine tRNA Phe, Thr12 ECF401 Phenylalanine tRNA Phe 12 ECF402 Phenylalanine tRNA Phe 12ECF403 Phenylalanine tRNA Phe 12 ECF403G45 Phenylalanine tRNA Lys 12ECF5 Phenylalanine tRNA Phe 12 ECFG73 Phenylalanine tRNA Phe, Gln 12

[0026] References (Ref.) for Table 1:

[0027] 1) Neidhard (Ed.) “Escherichia coli and Salmonella, Cellular andMolecular Biology” (2nd. Edition, ASM Press, Washington, D.C., USA,1996)

[0028] 2) Martin et al, RNA 2 (9): 919-927, 1996

[0029] 3) Normanly et al, Proceedings of the National Academy of ScienceUSA, 83 (17): 6548-6552, 1986

[0030] 4) Accession Number K01197

[0031] 5) Yarus et al, Proceedings of the National Academy of ScienceUSA, 77 (9): 5092-5096, 1990

[0032] 6) McClain et al, Proceedings of the National Academy of ScienceUSA, 87 (23): 9260-9264, 1990

[0033] 7) Raftery et al, Journal of Bacteriology 158 (3): 849-859, 1984

[0034] 8) Raftery et al, Journal of Molecular Biology 190 (3): 513-517,1986

[0035] 9) Komatsoulis und Abelson, Biochemistry 32 (29): 7435-7444, 1993

[0036] 10) Martin et al, Nucleic Acids Research 23(5): 779-784, 1995

[0037] 11) Normanly et al, Journal of Molecular Biology 213 (4):719-726, 1990

[0038] 12) McClain und Foss, Journal of Molecular Biology, 202 (4):697-709, 1988 TABLE 2 List of ochre suppressors Name of the suppressorAmino acid gene/allele Name of the tRNA incorporated Ref. supB GlutaminetRNA 1 Gln 1 supC Tyrosine tRNA 1 Tyr 1 supD Serine tRNA 3 Ser 1 supG(=supL, supN) Lysine tRNA Lys 1 supM (=supB15) Tyrosine tRNA 2 Tyr 1supU (=su8) Tryptophane tRNA Trp 1 supV Tryptophane tRNA Trp 1tRNA^(Glu)-Su_(oc)205 Glutamic acid tRNA Glu 2 suIII+ ochre TyrosinetRNA 1 Tyr 3 trpT177 Tryptophane tRNA Gln 4 Su79 (=trpT179) TryptophanetRNA Trp 5

[0039] References (Ref.) for Table 2:

[0040] 1) Neidhard (Ed.) “Escherichia coli and Salmonella, Cellular andMolecular Biology” (2nd. Edition, ASM Press, Washington, D.C., USA,1996)

[0041] 2) Raftery und Yarus, EMBO Journal 6 (5): 1499-1506, 1987

[0042] 3) Accession number K01197

[0043] 4) Raftery et al, Journal of Bacteriology 158 (3): 849-859, 1984

[0044] 5) Raftery et al, Journal of Molecular Biology 190 (3): 513-517,1986 TABLE 3 List of opal suppressors Name of the suppressor Amino acidgene/allele Name of the tRNA incorporated Ref. supT Glycine tRNA 1 Gly 1sumA Glycine tRNA 2 Gly 1 ims, mutA Glycine tRNA 3 Gly 1 supU (=su9)Tryptophane tRNA Trp 1 se1C Serine tRNA Selenocysteine 2 GLNA3U70Glutamine tRNA Gln 3 trpT176 Tryptophane tRNA Trp 4 ARG Arginine tRNAArg 5 ARGII Arginine tRNA Arg 5 LysA20 Lysine tRNA Arg 5

[0045] References (Ref.) for Table 3:

[0046] 1) Neidhard (Ed.) “Escherichia coli and Salmonella, Cellular andMolecular Biology” (2nd. Edition, ASM Press, Washington, D.C., USA,1996)

[0047] 2) Schon et al, Nucleic Acids Research 17 (18): 7159-7165, 1989

[0048] 3) Weygand-Durasevic et al, Journal of Molecular Biology 240 (2):111-118, 1994

[0049] 4) Raftery et al, Journal of Bacteriology 158 (3): 849-859, 1984

[0050] 5) McClain et al, Proceedings of the National Academy of ScienceUSA, 87 (23): 9260-9264, 1990

[0051] In a first aspect of the invention, it was found that aminoacid-, in particular L-threonine-producing bacteria of the speciesEscherichia coli which contain a stop codon chosen from the group amber,ochre and opal in the coding region of the rpoS gene, in particularwithin the region corresponding to position 2 to 314 of the amino acidsequence of the RpoS protein in accordance with SEQ ID No. 1 and 2respectively, are further improved in their power to produce amino acidswhen a suppressor tRNA chosen from the group amber suppressor, ochresuppressor and opal suppressor is incorporated therein. As a result ofthe measures according to the invention, the activity or concentrationof the RpoS protein or σ³⁸ factor is lowered in general to >0 to 75%,for example 1 to 75%, to >0 to 50%, for example 0.5 to 50%, to >0 to 25%for example 0.25 to 25%, to >0 to 10%, for example 0.1 to 10%, or to >0to 5%, for example 0.05 to 5% of the activity or concentration of thewild type protein, or of the activity or concentration of the protein inthe initial microogranism. The presence of the suppressor(s) mentionedprevents the activity of the RpoS proteins or σ³⁸ factor dropping rightdown to 0.

[0052] Accordingly, the invention provides a process for decreasing theintracellular activity or concentration of the RpoS proteins or σ³⁸factor in amino acid-producing bacteria of the familyEnterobacteriaceae, in particular of the species Escherichia coli,wherein in these bacteria 1) at least one stop codon chosen from thegroup amber, ochre and opal is incorporated in the coding region of therpoS gene, in particular within the region corresponding to position 2to 314 of the amino acid sequence of the RpoS protein in accordance withSEQ ID No. 1 or 2, and 2) the corresponding suppressor tRNA gene(s) orallele(s) which code(s) for the suppressor tRNA, chosen from the groupamber suppressor, ochre suppressor and opal suppressor, is/areincorporated in these bacteria.

[0053] The invention also provides a process for preparing aminoacid-producing bacteria of the family Enterobacteriaceae, in particularof the species Escherichia coli, wherein in these bacteria 1) a stopcodon chosen from the group amber, ochre and opal is incorporated in thecoding region of the rpoS gene, in particular within the regioncorresponding to position 2 to 314 of the amino acid sequence of theRpoS protein in accordance with SEQ ID No. 1 or 2, and that 2) asuppressor tRNA gene or allele which codes for a suppressor tRNA chosenfrom the group amber suppressor, ochre suppressor and opal suppressor isincorporated in these bacteria.

[0054] Finally, the invention provides amino acid-producing bacteria ofthe family Enterobacteriaceae, in particular of the species Escherichiacoli, which contain at least one stop codon chosen from the group amber,ochre and opal in the coding region of the rpoS gene, in particularwithin the region corresponding to position 2 to 314 of the amino acidsequence of the RpoS protein in accordance with SEQ ID No. 1 or 2, andcontain the corresponding suppressor tRNA gene or allele which codes forthe associated suppressor tRNA, chosen from the group amber suppressor,ochre suppressor and opal suppressor.

[0055] With regard to the coding region of the rpoS gene, the followingsegments have proven to be particularly advantageous for theincorporation of a stop codon chosen from the group amber, ochre andopal, preferably amber:

[0056] segment of the coding region between positions 2 and 95, forexample position 33, corresponding to the amino acid sequence of theRpoS protein, given in SEQ ID No. 1 or SEQ ID No. 2,

[0057] segment of the coding region between positions 99 and 168, forexample position 148, corresponding to the amino acid sequence of theRpoS protein, given in SEQ ID No. 1 or SEQ ID No. 2,

[0058] segment of the coding region between positions 190 and 245,corresponding to the amino acid sequence of the RpoS protein, given inSEQ ID No. 1 or SEQ ID No. 2,

[0059] segment of the coding region between positions 266 and 281, forexample position 270 corresponding to the amino acid sequence of theRpoS protein, given in SEQ ID No. 1 or SEQ ID No. 2, and

[0060] segment of the coding region between positions 287 and 314, forexample position 304, corresponding to the amino acid sequence of theRpoS protein, given in SEQ ID No. 1 or SEQ ID No. 2,

[0061] In the event that the coding region of the rpoS gene has a stopcodon of the amber type, an amber suppressor chosen from the group inTable 1 is preferably used.

[0062] In the event that the coding region of the rpoS gene has a stopcodon of the ochre type, an ochre suppressor chosen from the group inTable 2 is preferably used.

[0063] In the event that the coding region of the rpoS gene has a stopcodon of the opal type, an opal suppressor chosen from the group inTable 3 is preferably used.

[0064] Those amino acid-producing bacteria of the species Escherichiacoli which are particularly preferred are those which contain a stopcodon of the amber type at the point in the nucleotide sequencecorresponding to position 33 in the amino acid sequence of the RpoS geneproduct in accordance with SEQ ID NO. 1 or SEQ ID NO. 2 and preferablycontain an amber suppressor chosen from the group in Table 1, inparticular the suppressor supE or the suppressor supD, and which producethe amounts of L-lysine, compared with the amount of desired L-aminoacid, cited above.

[0065] Depending on the suppressor used, the bacteria form a RpoS geneproduct or σ³⁸ factor which contains, at position 33 of the amino acidsequence in accordance with SEQ ID NO. 2, instead of L-glutamine, anamino acid chosen from the group L-serine, L-tyrosine, L-leucine,L-tryptophane, L-lysine, L-alanine, L-arginine, L-phenylalanine,L-cysteine, L-proline, L-histidine, L-threonine and L-valine. Thus, forexample when using the suppressor supD, L-serine is incorporated insteadof L-glutamine. When using suppressors which incorporate the amino acidL-glutamine in position 33 of the RpoS gene product or σ³⁸ factor, suchas for example supE, the amino acid sequence is not altered.

[0066] L-threonine-producing bacteria of the species Escherichia coliwhich contain the rpoS allele given in SEQ ID NO. 3 and the suppressorsupE given in SEQ ID NO. 4 are very particularly preferred.

[0067] The invention also provides, corresponding to this first aspectof the invention, a process for preparing amino acids or aminoacid-containing feedstuffs additives in which the following steps areperformed:

[0068] a) fermentation of bacteria from the family Enterobacteriaceaewhich contain 1) at least one stop codon chosen from the group amber,ochre and opal, preferably amber, in the coding region of the rpoS gene,in particular within the region corresponding to position 2 to 314 ofthe amino acid sequence of the RpoS protein in accordance with SEQ IDNO. 1 or 2 and 2) the corresponding suppressor tRNA gene(s) chosen fromthe group amber suppressor, ochre suppressor and opal suppressor, in asuitable medium,

[0069] b) enrichment of the amino acid in the fermentation broth,

[0070] c) isolation of the amino acid or amino acid-containingfeedstuffs additive from the fermentation broth, optionally with

[0071] d) constituents from the fermentation broth and/or the biomass(≧0 to 100%).

[0072] A process for preparing amino acids or amino acid-containingfeedstuffs additives is preferred in which Enterobacteriaceae whichcontain a stop codon of the amber type in the coding region of the rpoSgene corresponding to position 33 of the amino acid sequence of the RpoSprotein in accordance with SEQ ID NO. 1 or 2 and an amber suppressor,preferably supE, are fermented in a suitable medium.

[0073] The feedstuffs additives according to the invention may befurther processed in the liquid and also in the solid form.

[0074] Mutations by means of which a stop codon is introduced into thereading frame of the rpoS gene can be produced directly in the relevanthost by classical mutagenesis methods using mutagenic substances suchas, for example, N-methyl-N′nitro-N-nitrosoguanidine, or ultravioletlight.

[0075] Furthermore, in vitro methods using isolated rpoS DNA such as,for example, treatment with hydroxylamine can be used for mutagenesis(J. H. Miller: A Short Course in Bacterial Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1992). Finally, processes forsite-oriented mutagenesis using mutagenic oligonucleotides (T. A. Brown:Gentechnologie für Einsteiger, Spektrum Akademischer Verlag, Heidelberg,1993) or the polymerase chain reaction (PCR), as is described in thebook by Newton and Graham (PCR, Spektrum Akademischer Verlag,Heidelberg, 1994), can be used. The mutations produced can be determinedand tested by DNA sequencing, for example using Sanger et al.'s method(Proceedings of the National Academy of Science USA 74 (12): 5463-5467(1977)).

[0076] Suitable mutations can be incorporated into the desired strainswith the aid of recombination processes by means of gene or allelereplacement. A commonly-used method is the method of gene replacementwith the aid of a conditional replicating pSC101 derivate pMAK705, asdescribed by Hamilton et al. (Journal of Bacteriology 171 (9): 4617-4622(1989)). Other methods described in the prior art such as, for example,the Martinez-Morales et al. method (Journal of Bacteriology 181 (22):7143-7148 (1999)) or the Boyd et al. method (Journal of Bacteriology 182(3): 842-847 (2000)) can also be used.

[0077] It is also possible to transfer mutations into the desiredstrains by conjugation or transduction.

[0078] Finally, it is possible to use alleles of the rpoS gene knownfrom the prior art which contain a stop codon in the reading frame andintroduce these into the desired strains using the methods describedabove.

[0079] The strains obtained in the way described above are preferablymutants, transformants, recombinants, transductants or transconjugants.

[0080] In order to produce suppressor mutations in tRNA genes, basicallythe same methods as described for the rpoS gene can be used. Methodsusing oligonucleotide engineering, such as were used, for example, byKhorana (Science 203(4381): 614-625 (1979)), can also be used.Furthermore, the tRNA suppressor genes used in the prior art may be usedin particular. Methods for searching for, characterising and determiningthe efficiency of tRNA suppressors are described in the prior art, forexample in Miller and Albertini (Journal of Molecular Biology 164 (1):59-71 (1983)), in McClain and Foss (Journal of Molecular Biology 202(4): 697-709 (1988)), in Normanly et al. (Journal of Molecular Biology213 (4): 719-726 (1990)), in Kleina et al. (Journal of Molecular Biology213: 705-717 (1990)), in Lesley et al. (Promega Notes Magazine 46, p.02. (1994)) and in Martin et al. (Nucleic Acids Research 23 (5): 779-784(1995)).

[0081] A second aspect of the invention relates to the variant of theRpoS protein given in SEQ ID NO. 6. While working on the presentinvention, the coding region of this variant of the RpoS protein wasidentified. This is given in SEQ ID NO. 5.

[0082] Accordingly, the invention provides Enterobacteriaceae, inparticular those which produce amino acids, which contain or form theRpoS protein given in SEQ ID NO. 6. It is also known that the N-terminalmethionine in the proteins formed can be split off by enzymes present inthe host.

[0083] Furthermore, the invention provides, corresponding to this secondaspect, a process for preparing amino acids or amino acid-containingfeedstuffs additives, in which the following steps are performed:

[0084] a) fermentation of Enterobacteriaceae which contain or form aRpoS protein with the amino acid sequence given in SEQ ID NO. 6,

[0085] b) enrichment of the amino acid in the fermentation broth,

[0086] c) isolation of the amino acid or amino acid-containingfeedstuffs additive from the fermentation broth, optionally with

[0087] d) constituents of the fermentation broth and/or the biomass (≧0to 100%).

[0088] Furthermore, for production of L-threonine with bacteria from thefamily Enterobacteriaceae, it may be advantageous in addition toincorporating a stop codon in the coding region of the rpoS gene and acorresponding suppressor for a stop codon or in addition to expressingthe variant of the RpoS protein given in SEQ ID NO. 6, to enhance one ormore enzymes from the known threonine biosynthesis pathway or enzyme(s)for anaplerotic metabolism or enzymes for the production of reducednicotinamide-adenine-dinucleotide phosphate or enzymes for glycolysis orPTS enzymes or enzymes for sulfur metabolism.

[0089] The expression “enhancement” in this connection describes anincrease in the intracellular activity or concentration of one or moreenzymes or proteins in a microorganism which are coded by thecorresponding DNA by, for example, increasing the copy number of thegene or genes, using a strong promoter or a gene which codes for acorresponding enzyme or protein with higher activity and optionallycombining these measures.

[0090] The use of endogenic genes is generally preferred. “Endogenicgenes” or “endogenic nucleotide sequences” are understood to be thegenes or nucleotide sequences which are present in the population of aspecies.

[0091] Using the measures of enhancement, in particular overexpression,the activity or concentration of the corresponding protein is generallyincreased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, at most up to 1000% or 2000%, with respect to the wild typeprotein or the activity or concentration of the protein in the initialmicroorganism.

[0092] Thus, for example, one or more genes, chosen from the followinggroup, may be enhanced, in particular overexpressed:

[0093] the thrABC operon coding for aspartate kinase, homoserinedehydrogenase, homoserine kinase and threonine synthase (U.S. Pat. No.4,278,765),

[0094] the pyc gene coding for pyruvate carboxylase (DE-A-19 831 609),

[0095] the pps gene coding for phosphoenolpyruvate synthase (Molecularand General Genetics 231 (2):332-336 (1992)),

[0096] the ppc gene coding for phosphoenolpyruvate carboxylase (Gene 31:279-283 (1984)),

[0097] the genes pntA and pntB coding for transhydrogenase (EuropeanJournal of Biochemistry 158: 647-653 (1986)),

[0098] the rhtB gene imparting homoserine resistance (EP-A-0 994 190),

[0099] the mqo gene coding for malate:quinone oxidoreductase (DE 100 34833.5),

[0100] the rhtC gene imparting threonine resistance (EP-A-1 013 765),

[0101] the thrE gene from Corynebacterium glutamicum coding forthreonine export protein (DE 100 264 94.8),

[0102] the gdhA gene coding for glutamate dehydrogenase (Nucleic AcidsResearch 11: 5257-5266 (1983); Gene 23: 199-209 (1983)),

[0103] the hns gene coding for DNA linkage protein HLP-II (Molecular andGeneral Genetics 212: 199-202 (1988)),

[0104] the pgm gene coding for phosphoglucomutase (Journal ofBacteriology 176: 5847-5851 (1994)),

[0105] the fba gene coding for fructose biphosphate aldolase(Biochemical Journal 257: 529-534 (1989)),

[0106] the ptsI gene from the ptsHIcrr operon, coding for enzyme I inthe phosphotransferase system (PTS) (Journal of Biological Chemistry262: 16241-16253 (1987)),

[0107] the ptsH gene from the ptsHIcrr operon, coding forphosphohistidine protein hexose phosphotransferase in thephosphotransferase system (PTS) (Journal of Biological Chemistry 262:16241-16253 (1987)),

[0108] the crr gene from the ptsHIcrr operon, coding for theglucose-specific IIA component in the phosphotransferase system (PTS)(Journal of Biological Chemistry 262: 16241-16253 (1987)),

[0109] the ptsG gene coding for the glucose-specific IIBC component inthe phosphotransferase system (PTS) (Journal of Biological Chemistry261: 16398-16403 (1986)),

[0110] the lrp gene coding for the regulator for the leucine regulon(Journal of Biological Chemistry 266: 10768-10774 (1991)),

[0111] the csrA gene coding for the global regulator (Journal ofBacteriology 175: 4744-4755 (1993)),

[0112] the fadR gene coding for the regulator for the fad regulon(Nucleic Acids Research 16: 7995-8009 (1988)),

[0113] the icIR gene coding for the regulator for central intermediarymetabolism (Journal of Bacteriology 172: 2642-2649 (1990)),

[0114] the mopB gene coding for the 10 Kd chaperone (Journal ofBiological Chemistry 261: 12414-12419 (1986)), which is also known bythe name groES,

[0115] the ahpC gene from the ahpCF operon, coding for the small subunitin alkyl hydroperoxide reductase (Proceedings of the National Academy ofSciences USA 92: 7617-7621 (1995))

[0116] the ahpF gene from the ahpCF operon, coding for the large subunitin alkyl hydroperoxide reductase (Proceedings of the National Academy ofSciences USA 92: 7617-7621 (1995))

[0117] the cysK gene coding for cysteine synthase A (Journal ofBacteriology 170: 3150-3157 (1988))

[0118] the cysB gene coding for the regulator for the cys regulon(Journal of Biological Chemistry 262: 5999-6005 (1987)),

[0119] the cysJ gene from the cysJIH operon, coding for flavoprotein inNADPH sulfite reductase (Journal of Biological Chemistry 264:15796-15808 (1989), Journal of Biological Chemistry 264: 15726-15737(1989)),

[0120] the cysH gene from the cysJIH operon, coding for adenylylsulfatereductase (Journal of Biological Chemistry 264: 15796-15808 (1989),Journal of Biological Chemistry 264: 15726-15737 (1989)), and

[0121] the cysI gene from the cysJIH operon, coding for haemoprotein inNADPH sulfite reductase (Journal of Biological Chemistry 264:15796-15808 (1989), Journal of Biological Chemistry 264: 15726-15737(1989)).

[0122] Furthermore, it may be advantageous for the production ofL-threonine with bacteria from the family Enterobacteriaceae, inaddition to incorporating a stop codon in the coding region of the rpoSgene and the corresponding suppressor for the stop codon, or in additionto expressing the variant of the RpoS protein given in SEQ ID NO. 6, toattenuate, in particular to switch off or to reduce the expression of,one or more genes chosen from the following group:

[0123] the tdh gene coding for threonine dehydrogenase (Ravnikar undSomerville; Journal of Bacteriology 169: 4716-4721 (1987)),

[0124] the mdh gene coding for malate dehydrogenase (E.C. 1.1.1.37)(Vogel et al.; Archives in Microbiology 149: 36-42 (1987)),

[0125] the gene product of the open reading frame (orf) yjfA (AccessionNumber AAC77180 for the National Center for Biotechnology Information(NCBI, Bethesda, Md., USA)),

[0126] the gene product of the open reading frame (orf) ytfP (AccessionNumber AAC77179 for the National Center for Biotechnology Information(NCBI, Bethesda, Md., USA)),

[0127] the pckA gene coding for the enzyme phosphoenolpyruvatecarboxykinase (Medina et al.; Journal of Bacteriology 172: 7151-7156(1990)),

[0128] the poxB gene coding for pyruvate oxidase (Grabau und Cronan;Nucleic Acids Research 14 (13): 5449-5460 (1986)),

[0129] the aceA gene coding for the enzyme isocitrate lyase (Matsuokound McFadden; Journal of Bacteriology 170: 4528-4536 (1988)),

[0130] the dgsA gene coding for the DgsA regulator in thephosphotransferase system (Hosono et al.; Bioscience, Biotechnology andBiochemistry 59: 256-261 (1995)), which is also known by the name mlcgene, and

[0131] the fruR gene coding for fructose repressor (Jahreis et al.;Molecular and General Genetics 226: 332-336 (1991)), which is also knownby the name cra gene.

[0132] The expression “attenuation” in this connection describes thedecrease in or switching off of the intracellular activity orconcentration of one or more enzymes or proteins in a microorganismwhich are coded by the corresponding DNA by, for example, using a weakpromoter or a gene or allele which codes for a corresponding enzyme witha low activity or inactivates the corresponding enzyme, protein or geneand optionally combining these measures. Using the measures ofattenuation, including decreasing the expression, the activity orconcentration of the corresponding protein is generally lowered to 0 to75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity orconcentration of the wild type protein, or the activity or concentrationof the protein in the initial microorganism. It has also beendemonstrated that in the case of the genes tdh, mdh, pckA, poxB, aceA,dgsA and fruR mentioned above and the open reading frames (ORF) yjfA andytfP and also for the genes ugpB (gene coding for periplastic linkageprotein in the sn-glycerine-3-phosphate transport system), aspA(aspartate ammonium lyase gene (=aspartase gene)), aceB (gene coding forthe enzyme malate synthase A) and aceK (gene coding for the enzymeisocitrate dehydrogenase kinase/phosphatase), attenuation can beachieved by 1) incorporating a stop codon, chosen from the group amber,ochre and opal, in the coding region of these genes and 2)simultaneously using a suppressor for the corresponding stop codon,chosen from the group amber suppressor, ochre suppressor and opalsuppressor. The use of the stop codon of the amber type and the ambersuppressor supE has proven to be particularly advantageous. Themethodology described can be transferred to any genes for whichattenuation or switching off is intended to be produced.

[0133] Microorganisms according to the invention can be cultivated in abatch process, in a fed batch process or in a repeated fed batchprocess. A review of known cultivation methods is given in the textbookby Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0134] The culture medium to be used has to satisfy in an appropriatemanner the demands of the particular strains. Descriptions of culturemedia for different microorganisms are given in the book “Manual ofMethods for General Bacteriology” by the American Society forBacteriology (Washington D.C., USA, 1981).

[0135] Sources of carbon which may be used are sugar and carbohydratessuch as e.g. glucose, saccharose, lactose, fructose, maltose, molasses,starch and optionally cellulose, oils and fats such as e.g. soy oil,sunflower oil, peanut oil and coconut fat, fatty acids such as e.g.palmitic acid, stearic acid and linoleic acid, alcohols such as e.g.glycerine and ethanol and organic acids such as e.g. acetic acid. Thesesubstances may be used individually or as mixtures.

[0136] Sources of nitrogen which may be used are organicnitrogen-containing compounds such as peptones, yeast extract, meatextract, malt extract, corn seep liquor, soy bean flour and urea orinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate. The sourcesof nitrogen may be used individually or as mixtures.

[0137] Sources of phosphorus which may be used are phosphoric acid,potassium dihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. The culture medium must alsocontain salts of metals, such as e.g. magnesium sulfate or iron sulfate,which are required for growth. Finally essential growth substances suchas amino acids and vitamins have to be used in addition to thesubstances mentioned above. Suitable precursors may also be added to theculture medium. The feedstocks mentioned may be added to the culture inthe form of a one-off portion or may be fed in a suitable manner duringcultivation.

[0138] To control the pH of the culture, basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or ammoniacal water or acidcompounds such as phosphoric acid or sulfuric acid are used in anappropriate manner. To control the development of foam, antifoamingagents such as e.g. fatty acid polyglycol esters are used. To maintainstability of the plasmids, suitable selectively acting substances suchas e.g. antibiotics may be added to the medium. In order to maintainaerobic conditions, oxygen or oxygen-containing gas mixtures such ase.g. air are introduced into the culture. The temperature of the cultureis normally 25° C. to 45° C. and is preferably 30° C. to 40° C.

[0139] The culture is continued until a maximum of L-amino acids hasbeen formed. This objective is normally achieved within 10 hours to 160hours.

[0140] Analysis of amino acids can be performed using anion exchangechromatography followed by ninhydrin derivatisation, as described inSpackman et al. (Analytical Chemistry 30: 1190-1206 (1958)), or it maybe performed using reversed phase HPLC, as described in Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

[0141] The following microorganism was deposited as a pure culture onSep. 9, 2002 at the German Collection of Microorganisms and CellCultures (DSMZ, Braunschweig, Germany) in accordance with the BudapestTreaty:

[0142]Escherichia coli strain DM1690 as DSM 15189. Strain DSM 15189contains a stop codon of the amber type at the point corresponding toposition 33 in the amino acid sequence of the RpoS protein and an ambersuppressor and produces threonine.

[0143] The process according to the invention is used for thefermentative preparation of L-threonine, L-isoleucine, L-methionine,L-homoserine, in particular L-threonine.

[0144] The present invention is explained in more detail in thefollowing, making use of working examples.

[0145] The minimal (M9) and universal media (LB) for E. coli aredescribed by J. H. Miller (A Short Course in Bacterial Genetics (1992),Cold Spring Harbor Laboratory Press). Isolation of plasmid DNA from E.coli and all techniques relating to restriction and Klenow and alkalinephosphatase treatment are performed as described by Sambrook et al.(Molecular Cloning. A Laboratory Manual (1989), Cold Spring HarborLaboratory Press). The transformation of E. coli, unless describeddifferently, is performed as described in Chung et al. (Proceedings ofthe National Academy of Sciences of the United States of America 86:2172-2175(1989)). P1 transductions are performed as described byLengeler et al. (Journal of Bacteriology 124: 26-38 (1975)).

EXAMPLE 1

[0146] Transduction of the scr Gene Locus into E. coli K12 Strain MG1655

[0147] The scr regulon in the naturally occurring plasmid pUR400 (Schmidet al., Molecular Microbiology 2: 1-8 (1988)) promotes the ability tomake use of saccharose as a source of carbon. With the aid of theplasmid pKJL710 (Ulmke et al., Journal of Bacteriology 181: 1920-1923(1999)), which contains the scr regulon between the two reverse sequencerepeats of the transposon Tnl721 (Ubben and Schmitt, Gene 41: 154-152(1986)), followed by transformation, transposition, conjugation andtransduction, the scr regulon can be transferred to the chromosome ofEscherichia coli K12. A strain called LJ210 contains the scr regulonintegrated in the chromosome at position 6 minutes according toBerlyn-Karte. The bacteriophage P1 is multiplied in this strain and E.coli K12 strain MG1655 (Guyer et al., Cold Spring Harbor Symp., Quant.Biology 45: 135-140 (1981)) is infected with the isolated phage lysate.By plating out on saccharose-containing (2 g/l) minimal medium, MG1655transductants are obtained which can use saccharose as a source ofcarbon. A selected clone is given the name MG1655scr.

EXAMPLE 2

[0148] In-Vivo Mutagenesis of the Strain MG1655scr

[0149] Starting from MG1655scr, after incubation at 37° C. in minimalagar to which has been added 2 g/l saccharose and 4 g/lDL-8-hydroxynorvaline (Sigma, Deisenhofen, Germany), spontaneous mutantsare isolated which are resistant to the threonine analogonα-amino-β-hydroxyvaleric acid (AHV). A selected mutant is given the nameMG1655scrAHVR1.

EXAMPLE 3

[0150] Incorporation of a Stop Codon Mutation in the rpoS Gene inMG1655scrAHVR1 by Site-Specific Mutagenesis

[0151] 3.1 Cloning the rpoS Gene from MG1655

[0152] The Escherichia coli strain MG1655 is used as donor forchromasomal DNA. A DNA fragment which contains the region of the rpoSgene to be mutated in the middle region is amplified using thepolymerase chain reaction (PCR) and synthetic oligonucleotides. Startingfrom the known sequence of the rpoS gene (Accession Number AE000358, SEQID No. 1) for Escherichia coli K12, the following primeroligonucleotides (MWG Biotech, Ebersberg, Germany) are chosen for PCR:rpoS9: 5′ CAGTTATAGCGGCAGTACC 3′ (SEQ ID No.7) rpoS4:5′ GGACAGTGTTAACGACCATTCTC 3′ (SEQ ID No.8)

[0153] The chromosomal E. coli K12 DNA is isolated in accordance withthe manufacturer's data using “Qiagen Genomic-tips 100/G” (Qiagen,Hilden, Germany). A roughly 2 kbp length DNA fragment can be isolatedusing the specific primers under standard PCR conditions (Innis et al.(1990) PCR Protocols. A Guide to Methods and Applications, AcademicPress) with vent polymerase (New England Biolabs GmbH, Frankfurt,Germany).

[0154] The amplified DNA fragment is identified using gelelectrophoresis in an agarose gel (0.8%) and purified with the QIAquickPCR Purification Kit (Qiagen, Hilden, Germany). The purified PCR productis ligated in accordance with the manufacturer's data using the vectorpCR-Blunt II-TOPO (Zero Blunt TOPO PCR Cloning Kit, Invitrogen,Groningen, Holland) and transformed in E. coli strain TOP10 (Invitrogen,Groningen, Holland). Selection of the plasmid-containing cells isperformed on LB agar to which has been added 50 mg/l kanamycin. Afterplasmid DNA isolation, the vector is tested using restriction cleavageand separation in agarose gel (0.8%). The amplified DNA fragment istested by sequence analysis. The sequence in the PCR product agrees withthe sequence given in SEQ ID NO. 9. The plasmid obtained is given thename pCRBluntrpoS9-4.

[0155] 3.2 Replacing a Glutamine Codon with an Amber Stop Codon bySite-Specific Mutagenesis

[0156] Site-directed mutagenesis is performed with the QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, USA). The followingprimer oligonucleotides are chosen for linear amplification: rpoSamber1:(SEQ ID No.10) 5′ GGCCTTAGTAGAA (TAG) GAACCCAGTGATAACG 3′ rpoSamber2:(SEQ ID No.11) 5′ CGTTATCACTGGGTTC (CTA) TTCTACTAAGGCC 3′

[0157] These primers are synthesised by MWG Biotech. The amber stopcodon which is meant to replace the glutamine at position 33 in theamino acid sequence is labelled with brackets in the nucleotidesequences shown above. The plasmid pCRBluntrpoS9-4 described in example3.1 is used with each of the two primers complementary to a strand inthe plasmid for linear amplification by means of PfuTurbo DNApolymerase. A mutated plasmid with broken circular strands is producedduring this elongation of the primer. The product from linearamplification is treated with DpnI. This endonuclease specifically cutsthe methylated and semi-methylated template DNA. The newly synthesisedbroken, mutated vector DNA is transformed in E. coli strain XL1 Blue(Bullock, Fernandez and Short, BioTechniques 5(4) 376379 (1987)). Aftertransformation, the XL1 Blue cells repair the breaks in the mutatedplasmids. Selection of the transformants is performed on LB medium with50 mg/l kanamycin. The plasmid obtained is tested after isolation of theDNA by means of restriction cleavage and separation in agarose gel(0.8%). The DNA sequence of the mutated DNA fragment is tested bysequence analysis. The sequence agrees with the sequence given in SEQ IDNO. 3 in the region of the rpoS gene. The plasmid obtained is given thename pCRBluntrpoSamber.

[0158] 3.3 Construction of the Replacement Vector pMAK705rpoSamber

[0159] The plasmid pCRBluntrpoSamber described in example 3.2 is cleavedwith restriction enzymes BamHI and XbaI (Gibco Life Technologies GmbH,Karlsruhe, Germany). After separation in an agarose gel (0.8%) theroughly 2.1 kbp length rpoS fragment containing the mutation is isolatedwith the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Theplasmid pMAK705 described in Hamilton et al. (Journal of Bacteriology171: 4617-4622 (1989)) is cleaved with restriction enzymes BamHI andXbaT and ligated with the isolated rpoS fragment (T4-DNA-Ligase,Amersham-Pharmacia, Freiburg, Germany). Then the E. coli strain DH5α(Grant et al.; Proceedings of the National Academy of Sciences USA 87:4645-4649 (1990)) is transformed with the ligation batch (Hanahan, In.DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Habor,N.Y., 1989). Selection of cells containing the plasmid is performed byplating out the transformation batch on LB agar (Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2^(nd) Ed., Cold Spring Habor,N.Y., 1989) which is supplemented with 20 mg/l chloramphenicol.

[0160] Plasmid DNA is isolated from a transformant using the QIAquickSpin Miniprep Kit from Qiagen and tested by restriction cleavage withthe enzymes BamHI, EcoRI, EcoRV, StuI and XbaI followed by agarose gelelectrophoresis. The plasmid is given the name pMAK705rpoSamber. A mapof the plasmid is shown in FIG. 1.

[0161] 3.4 Site-Specific Mutagenesis of the rpoS Gene E. coli StrainMG1655scrAHVR1

[0162] For site-specific mutagenesis of the rpoS gene, the strainMG1655scrAHVR1 described in example 2 is transformed with the plasmidpMAK705rpoSamber. Gene replacement is performed using the selectionprocess described in Hamilton et al. (Journal of Bacteriology 171:4617-4622 (1989)). Proof that mutation of the rpoSamber allele has takenplace in the chromosome is performed using the LightCycler from RocheDiagnostics (Mannheim, Germany). The LightCycler is a combinedinstrument consisting of a thermocycler and a fluorimeter.

[0163] In the first phase, a roughly 0.3 kbp length DNA section whichcontains the mutation site is amplified by means of PCR (Innis et al.,PCR Protocols. A Guide to Methods and Applications, 1990, AcademicPress) using the following primer oligonucleotides: rpoSLC1:5′ CGGAACCAGGCTTTTGCTTG 3′ (SEQ ID No.12) rpoSLC2:5′ GCGCGACGCGCAAAATAAAC 3′ (SEQ ID No.13)

[0164] In the second phase the presence of mutation is proven usingmelting curve analysis (Lay et al.; Clinical Chemistry 43: 2262-2267(1997)) with two additional oligonucleotides of different lengths andlabelled with different fluorescent dyes (LightCycler(LC)-Red640 andfluorescein) which hybridise in the region of the mutation site, usingthe “Fluorescence Resonance Energy Transfer” method (FRET). rpoSamberC:5′LC-Red640-CTTAGTAGAACAGGAACCCAGTG-(B) 3′ (SEQ ID No.14) rpoSamberA:5′ GATGAGAACGGAGTTGAGGTTTTTGACGAAAAGG-Fluorescein 3′ (SEQ ID No.15)

[0165] The primers shown for PCR are synthesised by MWG Biotech(Ebersberg, Germany) and the oligonucleotides for hybridisation aresynthesised by TIB MOLBIOL (Berlin, Germany).

[0166] In this way, a clone is identified which contains a thymidinebase instead of a cytosine base at position 952 in the DNA sequence forthe rpoS PCR product (SEQ ID No. 9). The base triplet thymine adenineguanine is present at position 97-99 of the coding sequence for the rpoSallele (SEQ ID No. 3) and codes for an amber stop codon which leads totermination of translation. This clone is given the nameMG1655scrAHVR1rpoS.

EXAMPLE 4

[0167] In-Vivo Selection of an Amber Suppressor Mutation in the StrainMG1655scrAHVR1rpoS

[0168] 4.1 Construction of a Vector with an Amber Stop Codon in the CatGene

[0169] For selection of a suppressor mutation, an amber stop codon isincorporated in the cat gene which codes for chloramphenicol acetyltransferase and imparts resistance to the antibiotic chloramphenicol.

[0170] For this purpose, the Cat gene block is ligated in the HindIIIlinearised vector pTrc99A (both from Pharmacia Biotech, Freiburg,Germany). The E. coli strain DH5α (Grant et al.; Proceedings of theNational Academy of Sciences USA 87: 4645-4649 (1990)) is transformedwith the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach.Vol. 1, ILR-Press, Cold Spring Habor, N.Y., 1989). Selection of cellscontaining the plasmid is performed by plating out the transformationbatch on LB agar (Sambrook et al., Molecular Cloning: A LaboratoryManual. 2^(nd) Ed., Cold Spring Habor, N.Y., 1989) which is supplementedwith 50 mg/l ampicillin. Plasmid DNA is isolated from a transformantusing the QIAquick Spin Miniprep Kit from Qiagen and tested byrestriction cleavage followed by agarose gel electrophoresis. Theplasmid is given the name pTrc99Acat.

[0171] To incorporate an amber stop codon in the coding region of thecat gene, starting from the sequence for the cat gene, primers whichcontain the cleavage sites for the restriction enzymes AccIII and MscIare synthesised by MWG Biotech, Ebersberg, Germany. The recognitionsites for the restriction enzymes are labelled by underscoring in thenucleotide sequences shown below. In the catAccIII primer, behind theAccIII cleavage site, two ATG codons which code for the amino acidmethionine at positions 75 and 77 in the cat protein are changed intoTAG codons. The amber codons are shown inside brackets in the nucleotidesequences given below. catAccIII: 5′ GCTCATCCGGAATTCCGT(TAG)GCA(TAG)AAAG 3′ (SEQ ID No.16) catMscI: 5′ GTCCATATTGGCCACGTTTAAATC3′ (SEQ ID No.17)

[0172] Plasmid DNA from the vector pTrc99Acat is used for PCR. A roughly300 bp length DNA fragment can be amplified with the specific primersunder standard PCR conditions (Innis et al. (1990) PCR Protocols. AGuide to Methods and Applications, Academic Press) using Pfu DNApolymerase (Promega Corporation, Madison, USA). The PCR product iscleaved with the restriction enzymes AccIII and MscI. The vectorpTrc99Acat is also digested with the enzymes AccIII and MscI, separatedin gel and a 4.7 kbp length fragment is isolated using the QIAquick GelExtraction Kit. The two fragments are ligated with T4 DNA ligase(Amersham-Pharmacia, Freiburg, Germany). The E. coli strain XL1-BlueMRF′ (Stratagene, La Jolla, USA) is transformed with the ligation batchand cells containing plasmid are selected on LB agar to which 50 μg/mlampicillin has been added. Successful cloning can be proven, afterplasmid DNA isolation, by control cleavage using the enzymes EcoRI,PvuII, SspI and StyI. The plasmid is given the name pTrc99Acatamber. Amap of the plasmid is shown in FIG. 2.

[0173] 4.2 Selection of Clones with an Amber Suppressor

[0174] The strain MG1655scrAHVR1rpoS described in example 3 istransformed with the vectors pTrc99Acat and pTrc99Acatamber. Selectionis performed on LB agar which has been supplemented with 20 or 50 μg/mlchioramphenicol. Chloramphenicol-resistant clones which have beentransformed with the vector pTrc99Acatamber are transferred to LB Agarto which 50 μg/ml ampicillin has been added, using a toothpick. PlasmidDNA is isolated from chloramphenicol and ampicillin-resistant clones.The vector pTrc99Acatamber is identified by restriction cleavage.

[0175] A chloramphenicol-resistant transformantMG1655scrAHVRrpoS/pTrc99Acatamber is cured by the plasmidpTrc99Acatamber by overinoculating several times in LB medium and isgiven the name DM1690.

[0176] 4.3 Identification of the Amber Suppressor Mutation in DM1690

[0177] 4.3.1 Testing for supD Mutation

[0178] A known mutation which leads to suppression of amber codons ispresent in the serU gene which codes for serine tRNA-2. The allele iscalled supD, the tRNA recognises amber codons and incorporates serine.In the sequence for the supD60 gene (Accession Number M10746) thecytosine adenine adenine anticodon in the wild type serU gene ismodified by base replacement to give cytosine thymine adenine and thusrecognises uracil adenine guanine codons.

[0179] A possible mutation in the chromasomal serU gene can be detectedusing the LightCycler from Roche Diagnostics (Mannheim, Germany). TheLightCycler is a combined instrument consisting of a thermocycler and afluorimeter.

[0180] In the first phase, a roughly 0.3 kbp length DNA sectioncontaining the mutation site is amplified by PCR (Innis et al., PCRProtocols. A Guide to Methods and Applications, 1990, Academic Press)using the following primer oligonucleotides: serULC1:5′ CTTGTACTTTCCAGGGCCAC 3′ (SEQ ID No.18) serULC2:5′ TTTACCAAAAGCAAGGCGGG 3′ (SEQ ID No.19)

[0181] In the second phase the presence of mutation is proven usingmelting curve analysis (Lay et al.; Clinical Chemistry 43: 2262-2267(1997)) with two additional oligonucleotides of different lengths andlabelled with different fluorescent dyes (LightCycler(LC)-Red640 andfluorescein) which hybridise in the region of the mutation site, usingthe “Fluorescence Resonance Energy Transfer” method (FRET). serUC:5′LC-Red640-TCCGGTTTTCGAGACCGGTC-(P) 3′ (SEQ ID No.20) serUA:5′ GAGGGGGATTTGAACCCCCGCTAGAGTTGCCCCTA-Fluorescein 3′ (SEQ ID No.21)

[0182] The primers for PCR shown are synthesised by MWG Biotech(Ebersberg, Germany) and the oligonucleotides for the hybridisationshown are synthesised by TIB MOLBIOL (Berlin, Germany).

[0183] It can be shown that the wild type serU gene is present in DM1690and thus there is no supD mutation.

[0184] 4.3.2 Sequence Analysis of the supE Allele in DM1690

[0185] Another known mutation which leads to suppression of amber codonsis present in the glnV gene which codes for glutamine tRNA-2. The alleleis called supE, the tRNA recognises amber codons and incorporatesglutamine. In the sequence for the glnV gene (Accession Number AE000170)the cytosine thymine adenine anticodon in the wild type glnV gene ismodified by base replacement to give cytosine thymine adenine and itrecognises uracil adenine guanine codons.

[0186] Starting from the known sequence for the glnV region ofEscherichia coli K12 (Accession Number AE000170), the following primeroligonucleotides (MWG Biotech, Ebersberg, Germany) are chosen for PCR:glnX1: 5′ CTGGCGTGTTGAAACGTCAG 3′ (SEQ ID No.22) glnX2:5′ CACGCTGTTCGCAACCTAACC 3′ (SEQ ID No.23)

[0187] The chromosomal DNA from DM1690 used for PCR is isolated inaccordance with the manufacturer's data using “Qiagen Genomic-tips100/G” (Qiagen, Hilden, Germany). A roughly 1 kpb length DNA fragmentcan be isolated using the specific primers under standard PCR conditions(Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications,Academic Press) with vent polymerase (New England Biolabs GmbH,Frankfurt, Germany). The PCR product is purified with the QIAquick PCRPurification Kit and sequenced by Qiagen Sequencing Services (QiagenGmbH, Hilden, Germany). The sequence obtained agrees with SEQ ID No. 4in the region of the glnV gene.

[0188] The strain DM1690 possesses the supE allele and suppresses ambercodons by incorporating glutamine.

EXAMPLE 5

[0189] Preparing L-Threonine Using the Strains MG1655scrAHVR1,MG1655scrAHVR1rpoS and DM1690

[0190] The strains MG1655scrAHVR1, MG1655scrAHVR1rpoS and DM1690 aremultiplied on minimal medium with the following composition: 3.5 g/lNa2HPO4*2H₂O, 1.5 g/l KH2PO4, 1 g/l NH4Cl, 0.1 g/l MgSO4*7H₂O, 2 g/lsaccharose, 20 g/l agar.

[0191] The cultures are incubated for 5 days at 37° C. The formation ofL-threonine is monitored in batch cultures of 10 ml which are containedin 100 ml Erlenmeyer flasks. For this purpose, 10 ml of preculturemedium with the following composition: 2 g/l yeast extract, 10 g/l(NH4)₂SO₄, 1 g/l KH2PO4, 0.5 g/l MgSO4*7H₂O, 15 g/l CaCO3, 20 g/lsaccharose, is inoculated and the mixture is incubated for 16 hours at37° C. and 180 rpm on an ESR incubator from Kühner AG (Birsfelden,Switzerland). 250 μl portions of this preculture in each case aretransferred to 10 ml of production medium (25 g/l (NH4)₂SO₄, 2 g/lKH2PO4, 1 g/l MgSO4*7H₂O, 0.03 g/l FeSO4*7H₂O, 0.018 g/l MnSO4*1H₂O, 30g/l CaCO3, 20 g/l saccharose) and incubated for 48 hours at 37° C. Afterincubation, the optical density (OD) of the culture suspension isdetermined using a LP2W photometer from Dr. Lange (Berlin, Germany) at ameasurement wavelength of 660 nm.

[0192] Then the concentration of L-threonine formed is determined in thesterile filtered supernatant liquid using an amino acid analyser fromEppendorf-BioTronik (Hamburg, Germany) by means of ion exchangechromatography and post-column reaction with ninhydrin detection.

[0193] Table 4 gives the results of the trial. TABLE 4 OD L-threonineStrain (660 nm) g/l MG1655scrAHVR1 5.6 2.15 MG1655scrAHVR1rpoS 5.3 2.34DM1690 5.2 2.46

BRIEF DESCRIPTION OF THE FIGURES

[0194]FIG. 1: pMAK705rpoSamber

[0195]FIG. 2: pTrc99Acatamber

[0196] Data relating to length are to be regarded as approximate data.The abbreviations and names used are defined as follows: cat:Chloramphenicol resistance gene rep-ts: Temperature-sensitivereplication region of the plasmid pSC101 rpoS: Coding region of the rpoSgene Amp: Ampicillin resistance gene lacI: Gene for the repressor genein the trc promoters Ptrc: trc promoter region, IPTG-inducible 5S: 5SrRNA region rrnBT: rRNA terminator region

[0197] The abbreviations for the restriction enzymes are defined asfollows: AccIII: Restriction endonuclease from Acinetobactercalcoaceticus BamHI: Restriction endonuclease from Bacillusamyloliquefaciens EcoRI: Restriction endonuclease from Escherichia coliEcoRV: Restriction endonuclease from Escherichia coli MscI: Restrictionendonuclease from Microcossus species PvuII: Restriction endonucleasefrom Proteus vulgaris SspI: Restriction endonuclease from Sphaerotilusspecies StuI: Restriction endonuclease from Streptomyces tubercidiusStyI: Restriction endonuclease from Salmonella typhi XbaI: Restrictionendonuclease from Xanthomonas badrii

[0198]

1 23 1 993 DNA Escherichia coli CDS (1)..(990) 1 atg agt cag aat acg ctgaaa gtt cat gat tta aat gaa gat gcg gaa 48 Met Ser Gln Asn Thr Leu LysVal His Asp Leu Asn Glu Asp Ala Glu 1 5 10 15 ttt gat gag aac gga gttgag gtt ttt gac gaa aag gcc tta gta gaa 96 Phe Asp Glu Asn Gly Val GluVal Phe Asp Glu Lys Ala Leu Val Glu 20 25 30 cag gaa ccc agt gat aac gatttg gcc gaa gag gaa ctg tta tcg cag 144 Gln Glu Pro Ser Asp Asn Asp LeuAla Glu Glu Glu Leu Leu Ser Gln 35 40 45 gga gcc aca cag cgt gtg ttg gacgcg act cag ctt tac ctt ggt gag 192 Gly Ala Thr Gln Arg Val Leu Asp AlaThr Gln Leu Tyr Leu Gly Glu 50 55 60 att ggt tat tca cca ctg tta acg gccgaa gaa gaa gtt tat ttt gcg 240 Ile Gly Tyr Ser Pro Leu Leu Thr Ala GluGlu Glu Val Tyr Phe Ala 65 70 75 80 cgt cgc gca ctg cgt gga gat gtc gcctct cgc cgc cgg atg atc gag 288 Arg Arg Ala Leu Arg Gly Asp Val Ala SerArg Arg Arg Met Ile Glu 85 90 95 agt aac ttg cgt ctg gtg gta aaa att gcccgc cgt tat ggc aat cgt 336 Ser Asn Leu Arg Leu Val Val Lys Ile Ala ArgArg Tyr Gly Asn Arg 100 105 110 ggt ctg gcg ttg ctg gac ctt atc gaa gagggc aac ctg ggg ctg atc 384 Gly Leu Ala Leu Leu Asp Leu Ile Glu Glu GlyAsn Leu Gly Leu Ile 115 120 125 cgc gcg gta gag aag ttt gac ccg gaa cgtggt ttc cgc ttc tca aca 432 Arg Ala Val Glu Lys Phe Asp Pro Glu Arg GlyPhe Arg Phe Ser Thr 130 135 140 tac gca acc tgg tgg att cgc cag acg attgaa cgg gcg att atg aac 480 Tyr Ala Thr Trp Trp Ile Arg Gln Thr Ile GluArg Ala Ile Met Asn 145 150 155 160 caa acc cgt act att cgt ttg ccg attcac atc gta aag gag ctg aac 528 Gln Thr Arg Thr Ile Arg Leu Pro Ile HisIle Val Lys Glu Leu Asn 165 170 175 gtt tac ctg cga acc gca cgt gag ttgtcc cat aag ctg gac cat gaa 576 Val Tyr Leu Arg Thr Ala Arg Glu Leu SerHis Lys Leu Asp His Glu 180 185 190 cca agt gcg gaa gag atc gca gag caactg gat aag cca gtt gat gac 624 Pro Ser Ala Glu Glu Ile Ala Glu Gln LeuAsp Lys Pro Val Asp Asp 195 200 205 gtc agc cgt atg ctt cgt ctt aac gagcgc att acc tcg gta gac acc 672 Val Ser Arg Met Leu Arg Leu Asn Glu ArgIle Thr Ser Val Asp Thr 210 215 220 ccg ctg ggt ggt gat tcc gaa aaa gcgttg ctg gac atc ctg gcc gat 720 Pro Leu Gly Gly Asp Ser Glu Lys Ala LeuLeu Asp Ile Leu Ala Asp 225 230 235 240 gaa aaa gag aac ggt ccg gaa gatacc acg caa gat gac gat atg aag 768 Glu Lys Glu Asn Gly Pro Glu Asp ThrThr Gln Asp Asp Asp Met Lys 245 250 255 cag agc atc gtc aaa tgg ctg ttcgag ctg aac gcc aaa cag cgt gaa 816 Gln Ser Ile Val Lys Trp Leu Phe GluLeu Asn Ala Lys Gln Arg Glu 260 265 270 gtg ctg gca cgt cga ttc ggt ttgctg ggg tac gaa gcg gca aca ctg 864 Val Leu Ala Arg Arg Phe Gly Leu LeuGly Tyr Glu Ala Ala Thr Leu 275 280 285 gaa gat gta ggt cgt gaa att ggcctc acc cgt gaa cgt gtt cgc cag 912 Glu Asp Val Gly Arg Glu Ile Gly LeuThr Arg Glu Arg Val Arg Gln 290 295 300 att cag gtt gaa ggc ctg cgc cgtttg cgc gaa atc ctg caa acg cag 960 Ile Gln Val Glu Gly Leu Arg Arg LeuArg Glu Ile Leu Gln Thr Gln 305 310 315 320 ggg ctg aat atc gaa gcg ctgttc cgc gag taa 993 Gly Leu Asn Ile Glu Ala Leu Phe Arg Glu 325 330 2330 PRT Escherichia coli 2 Met Ser Gln Asn Thr Leu Lys Val His Asp LeuAsn Glu Asp Ala Glu 1 5 10 15 Phe Asp Glu Asn Gly Val Glu Val Phe AspGlu Lys Ala Leu Val Glu 20 25 30 Gln Glu Pro Ser Asp Asn Asp Leu Ala GluGlu Glu Leu Leu Ser Gln 35 40 45 Gly Ala Thr Gln Arg Val Leu Asp Ala ThrGln Leu Tyr Leu Gly Glu 50 55 60 Ile Gly Tyr Ser Pro Leu Leu Thr Ala GluGlu Glu Val Tyr Phe Ala 65 70 75 80 Arg Arg Ala Leu Arg Gly Asp Val AlaSer Arg Arg Arg Met Ile Glu 85 90 95 Ser Asn Leu Arg Leu Val Val Lys IleAla Arg Arg Tyr Gly Asn Arg 100 105 110 Gly Leu Ala Leu Leu Asp Leu IleGlu Glu Gly Asn Leu Gly Leu Ile 115 120 125 Arg Ala Val Glu Lys Phe AspPro Glu Arg Gly Phe Arg Phe Ser Thr 130 135 140 Tyr Ala Thr Trp Trp IleArg Gln Thr Ile Glu Arg Ala Ile Met Asn 145 150 155 160 Gln Thr Arg ThrIle Arg Leu Pro Ile His Ile Val Lys Glu Leu Asn 165 170 175 Val Tyr LeuArg Thr Ala Arg Glu Leu Ser His Lys Leu Asp His Glu 180 185 190 Pro SerAla Glu Glu Ile Ala Glu Gln Leu Asp Lys Pro Val Asp Asp 195 200 205 ValSer Arg Met Leu Arg Leu Asn Glu Arg Ile Thr Ser Val Asp Thr 210 215 220Pro Leu Gly Gly Asp Ser Glu Lys Ala Leu Leu Asp Ile Leu Ala Asp 225 230235 240 Glu Lys Glu Asn Gly Pro Glu Asp Thr Thr Gln Asp Asp Asp Met Lys245 250 255 Gln Ser Ile Val Lys Trp Leu Phe Glu Leu Asn Ala Lys Gln ArgGlu 260 265 270 Val Leu Ala Arg Arg Phe Gly Leu Leu Gly Tyr Glu Ala AlaThr Leu 275 280 285 Glu Asp Val Gly Arg Glu Ile Gly Leu Thr Arg Glu ArgVal Arg Gln 290 295 300 Ile Gln Val Glu Gly Leu Arg Arg Leu Arg Glu IleLeu Gln Thr Gln 305 310 315 320 Gly Leu Asn Ile Glu Ala Leu Phe Arg Glu325 330 3 993 DNA Escherichia coli Allele (1)..(990) rpoS-Allele 3atgagtcaga atacgctgaa agttcatgat ttaaatgaag atgcggaatt tgatgagaac 60ggagttgagg tttttgacga aaaggcctta gtagaatagg aacccagtga taacgatttg 120gccgaagagg aactgttatc gcagggagcc acacagcgtg tgttggacgc gactcagctt 180taccttggtg agattggtta ttcaccactg ttaacggccg aagaagaagt ttattttgcg 240cgtcgcgcac tgcgtggaga tgtcgcctct cgccgccgga tgatcgagag taacttgcgt 300ctggtggtaa aaattgcccg ccgttatggc aatcgtggtc tggcgttgct ggaccttatc 360gaagagggca acctggggct gatccgcgcg gtagagaagt ttgacccgga acgtggtttc 420cgcttctcaa catacgcaac ctggtggatt cgccagacga ttgaacgggc gattatgaac 480caaacccgta ctattcgttt gccgattcac atcgtaaagg agctgaacgt ttacctgcga 540accgcacgtg agttgtccca taagctggac catgaaccaa gtgcggaaga gatcgcagag 600caactggata agccagttga tgacgtcagc cgtatgcttc gtcttaacga gcgcattacc 660tcggtagaca ccccgctggg tggtgattcc gaaaaagcgt tgctggacat cctggccgat 720gaaaaagaga acggtccgga agataccacg caagatgacg atatgaagca gagcatcgtc 780aaatggctgt tcgagctgaa cgccaaacag cgtgaagtgc tggcacgtcg attcggtttg 840ctggggtacg aagcggcaac actggaagat gtaggtcgtg aaattggcct cacccgtgaa 900cgtgttcgcc agattcaggt tgaaggcctg cgccgtttgc gcgaaatcct gcaaacgcag 960gggctgaata tcgaagcgct gttccgcgag taa 993 4 75 DNA Escherichia coli tRNA(1)..(75) supE-Allele 4 tggggtatcg ccaagcggta aggcaccgga ttctaattccggcattccga ggttcgaatc 60 ctcgtacccc agcca 75 5 714 DNA Escherichia coliCDS (1)..(711) 5 atg atc gag agt aac ttg cgt ctg gtg gta aaa att gcc cgccgt tat 48 Met Ile Glu Ser Asn Leu Arg Leu Val Val Lys Ile Ala Arg ArgTyr 1 5 10 15 ggc aat cgt ggt ctg gcg ttg ctg gac ctt atc gaa gag ggcaac ctg 96 Gly Asn Arg Gly Leu Ala Leu Leu Asp Leu Ile Glu Glu Gly AsnLeu 20 25 30 ggg ctg atc cgc gcg gta gag aag ttt gac ccg gaa cgt ggt ttccgc 144 Gly Leu Ile Arg Ala Val Glu Lys Phe Asp Pro Glu Arg Gly Phe Arg35 40 45 ttc tca aca tac gca acc tgg tgg att cgc cag acg att gaa cgg gcg192 Phe Ser Thr Tyr Ala Thr Trp Trp Ile Arg Gln Thr Ile Glu Arg Ala 5055 60 att atg aac caa acc cgt act att cgt ttg ccg att cac atc gta aag240 Ile Met Asn Gln Thr Arg Thr Ile Arg Leu Pro Ile His Ile Val Lys 6570 75 80 gag ctg aac gtt tac ctg cga acc gca cgt gag ttg tcc cat aag ctg288 Glu Leu Asn Val Tyr Leu Arg Thr Ala Arg Glu Leu Ser His Lys Leu 8590 95 gac cat gaa cca agt gcg gaa gag atc gca gag caa ctg gat aag cca336 Asp His Glu Pro Ser Ala Glu Glu Ile Ala Glu Gln Leu Asp Lys Pro 100105 110 gtt gat gac gtc agc cgt atg ctt cgt ctt aac gag cgc att acc tcg384 Val Asp Asp Val Ser Arg Met Leu Arg Leu Asn Glu Arg Ile Thr Ser 115120 125 gta gac acc ccg ctg ggt ggt gat tcc gaa aaa gcg ttg ctg gac atc432 Val Asp Thr Pro Leu Gly Gly Asp Ser Glu Lys Ala Leu Leu Asp Ile 130135 140 ctg gcc gat gaa aaa gag aac ggt ccg gaa gat acc acg caa gat gac480 Leu Ala Asp Glu Lys Glu Asn Gly Pro Glu Asp Thr Thr Gln Asp Asp 145150 155 160 gat atg aag cag agc atc gtc aaa tgg ctg ttc gag ctg aac gccaaa 528 Asp Met Lys Gln Ser Ile Val Lys Trp Leu Phe Glu Leu Asn Ala Lys165 170 175 cag cgt gaa gtg ctg gca cgt cga ttc ggt ttg ctg ggg tac gaagcg 576 Gln Arg Glu Val Leu Ala Arg Arg Phe Gly Leu Leu Gly Tyr Glu Ala180 185 190 gca aca ctg gaa gat gta ggt cgt gaa att ggc ctc acc cgt gaacgt 624 Ala Thr Leu Glu Asp Val Gly Arg Glu Ile Gly Leu Thr Arg Glu Arg195 200 205 gtt cgc cag att cag gtt gaa ggc ctg cgc cgt ttg cgc gaa atcctg 672 Val Arg Gln Ile Gln Val Glu Gly Leu Arg Arg Leu Arg Glu Ile Leu210 215 220 caa acg cag ggg ctg aat atc gaa gcg ctg ttc cgc gag taa 714Gln Thr Gln Gly Leu Asn Ile Glu Ala Leu Phe Arg Glu 225 230 235 6 237PRT Escherichia coli 6 Met Ile Glu Ser Asn Leu Arg Leu Val Val Lys IleAla Arg Arg Tyr 1 5 10 15 Gly Asn Arg Gly Leu Ala Leu Leu Asp Leu IleGlu Glu Gly Asn Leu 20 25 30 Gly Leu Ile Arg Ala Val Glu Lys Phe Asp ProGlu Arg Gly Phe Arg 35 40 45 Phe Ser Thr Tyr Ala Thr Trp Trp Ile Arg GlnThr Ile Glu Arg Ala 50 55 60 Ile Met Asn Gln Thr Arg Thr Ile Arg Leu ProIle His Ile Val Lys 65 70 75 80 Glu Leu Asn Val Tyr Leu Arg Thr Ala ArgGlu Leu Ser His Lys Leu 85 90 95 Asp His Glu Pro Ser Ala Glu Glu Ile AlaGlu Gln Leu Asp Lys Pro 100 105 110 Val Asp Asp Val Ser Arg Met Leu ArgLeu Asn Glu Arg Ile Thr Ser 115 120 125 Val Asp Thr Pro Leu Gly Gly AspSer Glu Lys Ala Leu Leu Asp Ile 130 135 140 Leu Ala Asp Glu Lys Glu AsnGly Pro Glu Asp Thr Thr Gln Asp Asp 145 150 155 160 Asp Met Lys Gln SerIle Val Lys Trp Leu Phe Glu Leu Asn Ala Lys 165 170 175 Gln Arg Glu ValLeu Ala Arg Arg Phe Gly Leu Leu Gly Tyr Glu Ala 180 185 190 Ala Thr LeuGlu Asp Val Gly Arg Glu Ile Gly Leu Thr Arg Glu Arg 195 200 205 Val ArgGln Ile Gln Val Glu Gly Leu Arg Arg Leu Arg Glu Ile Leu 210 215 220 GlnThr Gln Gly Leu Asn Ile Glu Ala Leu Phe Arg Glu 225 230 235 7 19 DNAEscherichia coli 7 cagttatagc ggcagtacc 19 8 23 DNA Escherichia coli 8ggacagtgtt aacgaccatt ctc 23 9 2012 DNA Escherichia coli 9 cagttatagcggcagtacct ataccgtgaa aaaaggcgac acacttttct atatcgcctg 60 gattactggcaacgatttcc gtgaccttgc tcagcgcaac aatattcagg caccatacgc 120 gctgaacgttggtcagacct tgcaggtggg taatgcttcc ggtacgccaa tcactggcgg 180 aaatgccattacccaggccg acgcagcaga gcaaggagtt gtgatcaagc ctgcacaaaa 240 ttccaccgttgctgttgcgt cgcaaccgac aattacgtat tctgagtctt cgggtgaaca 300 gagtgctaacaaaatgttgc cgaacaacaa gccaactgcg accacggtca cagcgcctgt 360 aacggtaccaacagcaagca caaccgagcc gactgtcagc agtacatcaa ccagtacgcc 420 tatctccacctggcgctggc cgactgaggg caaagtgatc gaaacctttg gcgcttctga 480 ggggggcaacaaggggattg atatcgcagg cagcaaagga caggcaatta tcgcgaccgc 540 agatggccgcgttgtttatg ctggtaacgc gctgcgcggc tacggtaatc tgattatcat 600 caaacataatgatgattacc tgagtgccta cgcccataac gacacaatgc tggtccggga 660 acaacaagaagttaaggcgg ggcaaaaaat agcgaccatg ggtagcaccg gaaccagttc 720 aacacgcttgcattttgaaa ttcgttacaa ggggaaatcc gtaaacccgc tgcgttattt 780 gccgcagcgataaatcggcg gaaccaggct tttgcttgaa tgttccgtca agggatcacg 840 ggtaggagccaccttatgag tcagaatacg ctgaaagttc atgatttaaa tgaagatgcg 900 gaatttgatgagaacggagt tgaggttttt gacgaaaagg ccttagtaga acaggaaccc 960 agtgataacgatttggccga agaggaactg ttatcgcagg gagccacaca gcgtgtgttg 1020 gacgcgactcagctttacct tggtgagatt ggttattcac cactgttaac ggccgaagaa 1080 gaagtttattttgcgcgtcg cgcactgcgt ggagatgtcg cctctcgccg ccggatgatc 1140 gagagtaacttgcgtctggt ggtaaaaatt gcccgccgtt atggcaatcg tggtctggcg 1200 ttgctggaccttatcgaaga gggcaacctg gggctgatcc gcgcggtaga gaagtttgac 1260 ccggaacgtggtttccgctt ctcaacatac gcaacctggt ggattcgcca gacgattgaa 1320 cgggcgattatgaaccaaac ccgtactatt cgtttgccga ttcacatcgt aaaggagctg 1380 aacgtttacctgcgaaccgc acgtgagttg tcccataagc tggaccatga accaagtgcg 1440 gaagagatcgcagagcaact ggataagcca gttgatgacg tcagccgtat gcttcgtctt 1500 aacgagcgcattacctcggt agacaccccg ctgggtggtg attccgaaaa agcgttgctg 1560 gacatcctggccgatgaaaa agagaacggt ccggaagata ccacgcaaga tgacgatatg 1620 aagcagagcatcgtcaaatg gctgttcgag ctgaacgcca aacagcgtga agtgctggca 1680 cgtcgattcggtttgctggg gtacgaagcg gcaacactgg aagatgtagg tcgtgaaatt 1740 ggcctcacccgtgaacgtgt tcgccagatt caggttgaag gcctgcgccg tttgcgcgaa 1800 atcctgcaaacgcaggggct gaatatcgaa gcgctgttcc gcgagtaagt aagcatctgt 1860 cagaaaggccagtctcaagc gaggctggcc ttttctgtgc acaataaaag gtccgatgcc 1920 catcggacctttttattaag gtcaaattac cgcccatacg caccaggtaa ttaagaatcc 1980 ggtaaaaccgagaatggtcg ttaacactgt cc 2012 10 31 DNA Artificial sequence rpoSamber1,modified Escherichia coli sequence 10 ggccttagta gaataggaac ccagtgataa c31 11 31 DNA Artificial sequence rpoSamber2, modified Escherichia colisequence 11 gttatcactg ggttcctatt ctactaaggc c 31 12 20 DNA Escherichiacoli 12 cggaaccagg cttttgcttg 20 13 20 DNA Escherichia coli 13gcgcgacgcg caaaataaac 20 14 23 DNA Artificial sequence rpoSamberC,modified Escherichia coli sequence 14 cttagtagaa caggaaccca gtg 23 15 34DNA Artificial sequence rpoSamberA, modified Escherichia coli sequence15 gatgagaacg gagttgaggt ttttgacgaa aagg 34 16 31 DNA Artificialsequence catAccIII, modified Escherichia coli sequence 16 gctcatccggaattccgtta ggcatagaaa g 31 17 24 DNA Artificial sequence catMscI,modified Escherichia coli sequence 17 gtccatattg gccacgttta aatc 24 1820 DNA Escherichia coli 18 cttgtacttt ccagggccac 20 19 20 DNAEscherichia coli 19 tttaggaaaa gcaaggcggg 20 20 20 DNA Artificialsequence serUC, modified Escherichia coli sequence 20 tccggttttcgagaccggtc 20 21 35 DNA Artificial sequence serUA, modified Escherichiacoli sequence 21 gagggggatt tgaacccccg gtagagttgc cccta 35 22 20 DNAEscherichia coli 22 ctggcgtgtt gaaacgtcag 20 23 21 DNA Escherichia coli23 cacgctgttc gcaacctaac c 21

1. Amino acid-producing bacteria from the family Enterobacteriaceae, inparticular from the species Escherichia coli, wherein these contain 1)within the coding region of the rpoS gene at least one (1) stop codonchosen from the group amber, ochre and opal and 2) the correspondingsuppressor(s) chosen from the group amber suppressor, ochre suppressorand opal suppressor.
 2. Amino acid-producing bacteria as claimed inclaim 1, wherein these contain within the coding region of the rpoS geneat least one (1) stop codon of the amber type and at least one (1) ambersuppressor.
 3. Amino acid-producing bacteria as claimed in claim 2,wherein the stop codon of the amber type lies within the coding regionof the rpoS gene corresponding to positions 2 to 314 in the amino acidsequence of the RpoS protein, in accordance with SEQ ID No.
 2. 4. Aminoacid-producing bacteria as claimed in claim 3, wherein the stop codon ofthe amber type lies within the coding region of the rpoS genecorresponding to positions 2 to 95 in the amino acid sequence of theRpoS protein, in accordance with SEQ ID No.
 2. 5. Amino acid-producingbacteria as claimed in claim 4, wherein the stop codon of the amber typelies within the coding region of the rpoS gene corresponding to position33 in the amino acid sequence of the RpoS protein, in accordance withSEQ ID No.
 2. 6. Amino acid-producing bacteria as claimed in claim 5,wherein the bacteria contain at least one (1) amber suppressor chosenfrom the group supD and supE.
 7. Amino acid-producing bacteria asclaimed in claim 6, wherein the bacteria contain at least the ambersuppressor supE.
 8. Amino acid-producing bacteria as claimed in claim 1,wherein they produce not more than 40% of L-lysine as a secondaryproduct, compared with the amount of desired L-amino acid.
 9. Aminoacid-producing bacteria as claimed in claim 8, wherein they produce notmore than 10% of L-lysine as a secondary product, compared with theamount of desired L-amino acid.
 10. Amino acid-producing bacteria asclaimed in claim 8, wherein they produce not more than 5% of L-lysine asa secondary product, compared with the amount of desired L-amino acid.11. Amino acid-producing bacteria as claimed in claim 1, wherein theamino acid is an amino acid chosen from the group L-threonine,L-isoleucine, L-homoserine, L-methionine, L-glutamic acid, L-valine andL-tryptophane.
 12. Amino acid-producing bacteria as claimed in claim 1,wherein the amino acid is L-threonine,
 13. L-threonine-producingbacteria from the family Enterobacteriaceae, in particular from thespecies Escherichia coli, wherein these contain a stop codon of theamber type within the coding region of the rpoS gene corresponding toposition 33 in the amino acid sequence of the RpoS protein, inaccordance with SEQ ID NO. 2, and the amber suppressor supE. 14.L-threonine-producing bacteria as claimed in claim 13, wherein one ormore of the genes chosen from the group given below are simultaneouslyenhanced, in particular overexpressed: 14.1 the thrABC operon coding foraspartate kinase, homoserine dehydrogenase, homoserine kinase andthreonine synthase, 14.2 the pyc gene coding for pyruvate carboxylase,14.3 the pps gene coding for phosphoenolpyruvate synthase, 14.4 the ppcgene coding for phosphoenolpyruvate carboxylase, 14.5 the pntA and pntBgenes coding for transhydrogenase, 14.6 the rhtB gene impartinghomoserine resistance, 14.7 the mqo gene coding for malate:quinoneoxidoreductase, 14.8 the rhtC gene imparting threonine resistance, 14.9the thrE gene from Corynebacterium glutamicum coding for threonineexport protein, 14.10 the gdhA gene coding for glutamate dehydrogenase,14.11 the hns gene coding for DNA linkage protein HLP-II, 14.12 the pgmgene coding for phosphoglucomutase, 14.13 the fba gene coding forfructose biphosphate aldolase, 14.14 the ptsi gene in the ptsHIcrroperon, coding for enzyme I in the phosphotransferase system (PTS),14.15 the ptsH gene in the ptsHIcrr operon, coding for phosphohistidineprotein hexose phosphotransferase in the phosphotransferase system(PTS), 14.16 the crr gene in the ptsHIcrr operon, coding for theglucose-specific IIA component in the phosphotransferase system (PTS),14.17 the ptsG gene coding for the glucose-specific IIBC component inthe phosphotransferase system (PTS), 14.18 the lrp gene coding for theregulator in the leucine regulon, 14.19 the csrA gene coding for theglobal regulator, 14.20 the fadR gene coding for the regulator in thefad regulon, 14.21 the iclR gene coding for the regulator in centralintermediary metabolism, 14.22 the mopB gene coding for the 10 Kdchaperone, which is also known by the name groES, 14.23 the ahpC gene inthe ahpCF operon, coding for the small subunit of alkyl hydroperoxidereductase, 14.24 the ahpF gene in the ahpCF operon, coding for the largesubunit of alkyl hydroperoxide reductase, 14.25 the cysK gene coding forcysteine synthase A, 14.26 the cysB gene coding for the regulator in thecys regulon, 14.27 the cysj gene in the cysJIH operon, coding forflavoprotein in NADPH sulfite reductase, 14.28 the cysH gene in thecysJIH operon, coding for adenylylsulfate reductase, and 14.29 the cysigene in the cysJIH operon, coding for haemoprotein in NADPH sulfitereductase.
 15. Amino acid-producing bacteria as claimed in claim 13,wherein other genes are attenuated.
 16. A process for decreasing theintracellular activity of RpoS protein or σ³⁸ factor in bacteria in thefamily Enterobacteriaceae, in particular in the species Escherichiacoli, wherein 1) at least one stop codon chosen from the group amber,ochre and opal, is incorporated in the coding region of the rpoS geneand 2) a corresponding suppressor tRNA gene or allele which codes for asuppressor tRNA chosen from the group amber suppressor, ochre suppressorand opal suppressor is incorporated.
 17. A process as claimed in claim16, wherein the activity or concentration of the RpoS protein or σ³⁸factor is lowered to >0 to 75%.
 18. A process as claimed in claim 16,wherein the activity or concentration of the RpoS protein or σ³⁸ factoris lowered to >0 to 5%.
 19. Amino acid-producing bacteria from thefamily Enterobacteriaceae, in particular from the species Escherichiacoli, which form a RpoS protein or σ³⁸ factor which contains, atposition 33 in the amino acid sequence in accordance with SEQ ID NO. 2,an amino acid chosen from the group L-serine, L-tyrosine, L-leucine,L-tryptophane, L-lysine, L-alanine, L-arginine, L-phenylalanine,L-cysteine, L-proline, L-histidine, L-threonine and L-valine, whereinthe bacteria produce one or more amino acids chosen from the groupL-threonine, L-isoleucine, L-homoserine, L-methionine, L-glutamic acid,L-valine and L-tryptophane.
 20. A process for preparing amino acids orfoodstuffs additives containing these which contains the following stepsa) fermentation of bacteria from the family Enterobacteriaceae, inparticular from the species Escherichia coli which 1) within the codingregion of the rpoS gene contain at least one stop codon chosen from thegroup amber, ochre and opal and 2) possess the correspondingsuppressor(s) chosen from the group amber suppressor, ochre suppressorand opal suppressor, b) enrichment of the amino acid in the medium or inthe cells of the microorganisms, and c) isolation of the amino acid,wherein optionally constituents from the fermentation broth and/or thebiomass in their entirety, or a proportion thereof (≧0 bis 100), remainin the product.
 21. A process as claimed in claim 20, wherein the aminoacid is an amino acid chosen from the group L-threonine, L-isoleucine,L-homoserine, L-methionine, L-glutamic acid, L-valine and L-tryptophane.22. A process as claimed in claim 21, wherein the amino acid isL-threonine.
 23. A process for preparing L-threonine which contains thefollowing steps a) fermentation of bacteria from the familyEnterobacteriaceae, in particular from the species Escherichia coliwhich 1) within the coding region of the rpoS gene corresponding toposition 33 in the amino acid sequence in accordance with SEQ ID No. 2contain at least one stop codon of the amber type and 2) possess atleast the corresponding amber suppressor supE, b) enrichment of theL-threonine in the medium or in the cells of the microorganisms, and c)isolation of the L-threonine, wherein optionally constituents from thefermentation broth and/or the biomass in their entirety, or a proportionthereof (≧0 bis 100), remain in the product.
 24. Amino acid-producingbacteria from the family Enterobacteriaceae, in particular from thespecies Escherichia coli, which contain or form the RpoS protein givenin SEQ ID NO.
 6. 25. A process for preparing amino acids or feedstuffsadditives containing amino acids in which the following steps areperformed: a) fermentation of Enterobacteriaceae which contain or form aRpoS protein with the amino acid sequence given in SEQ ID NO. 6, b)enrichment of the amino acid in the fermentation broth, c) isolation ofthe amino acid or amino acid-containing feedstuffs additive from thefermentation broth, optionally with d) constituents from thefermentation broth and/or the biomass (≧0 bis 100%).
 26. Escherichiacoli strain DM1690 deposited as DSM 15189 at the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).
 27. Aprocess as claimed in claim 23, wherein E. coli strain DM1690, depositedas DSM 15189, is used.
 28. A process for attenuating or switching offone or more of the genes or open reading frames in the familyEnterobacteriaceae, in particular in the species Escherichia coli,chosen from the group tdh, mdh, pckA, poxB, aceA, dgsA, fruR, ugpB,aspA, aceB, acek, yjfA and ytfP, wherein 1) at least one stop codonchosen from the group amber, ochre and opal is incorporated in thecoding region of the gene involved and 2) at the same time thecorresponding suppressor(s) chosen from the group amber suppressor,ochre suppressor and opal suppressor are incorporated.